Project Summary The goal of this project is to understand how the gating of HCN channels is regulated by domains located in the cytoplasmic, intracellular portion of the protein. HCN channels regulate pacemaking in the heart, and neuronal excitability in the brain. They have the general structure of voltage-gated K+ channels, but are activated upon membrane hyperpolarization and conduct an inward, depolarizing current. Importantly, their gating is modulated by the direct binding of cAMP to an intracellular cyclic-nucleotide binding domain (CNBD) located in the C-terminal portion of the protein. Recent structural data have also revealed the presence of a second, highly structured domain in the intracellular N-terminal portion of the protein, immediately preceding the first transmembrane domain (HCN domain). In this study, we will use a combination of structural and functional approaches to determine the potential role of the HCN domain in the modulation of channel gating, and the nature of the interactions between the C-terminal CNBD and N-terminal HCN domain. As different HCN isoforms (HCN1-4) exhibit markedly distinct biophysical properties, we will exploit these differences to identify key contacts and residues that may contribute to modulate the gating of HCN channels. In Aim 1, we will use cryo-electron microscopy (cryoEM) to determine the structure of the HCN4 isoform, in the presence and absence of cAMP, and compare its features to the available cryoEM structure of HCN1. In Aim 2, we will resolve the structure of HCN4 in the presence of ligands that interfere with the cAMP-mediated facilitation of channel opening (auxiliary subunit TRIP8b, biphenylurea compound BPU), and use the structural data acquired through Aims 1 and 2 to inform functional experiments designed to test and interpret any inferred model of structure/function relation. Finally, in Aim 3, we will use a similar combination of cryoEM and functional studies to study select HCN1 and HCN4 mutations found in patients with genetic epilepsy and cardiac arrhythmias.